To understand a biological process at the molecular level, it is important to be able to identify genes that are differentially expressed in response to a stimulus. Some differentially expressed genes have been identified by isolating total mRNA before and after exposure to a stimulus, and identifying particular mRNAs that are present at different levels in the two mRNA pools. However, many such genes remain undetected because these pools of total mRNA contain mostly uninformative transcripts, due to the relatively long half-lives of most mRNAs (on the order of hours) within terminally differentiated eukaryotic cells.
Various differential and subtractive methods have been employed to facilitate the discovery of new differentially expressed genes. However, these techniques usually require considerable effort. In an attempt to facilitate the isolation of newly synthesized RNAs, phosphothioate analogues of nucleoside triphosphates have been employed (see, e.g., Melvin et al., Eur. J Biochem. 92:373-379, 1978; Woodford et al., Anal. Biochem. 171:166-172, 1988; and Ono and Kawakami, J Biochem. 81:1247-1252, 1977). Following incubation with such analogues, newly synthesized mRNAs can be isolated by organomercury affinity chromatography. Such techniques can permit the isolation of newly synthesized RNA, but the analogues can be toxic and can reduce the efficiency of transcription.
Accordingly, there is a need in the art for improved methods for preferentially isolating newly synthesized mRNAs, and for evaluating changes in gene expression as cells or tissues respond to stimuli. The present invention fulfills these needs and further provides other related advantages.